SupportBiology of Inflammation/Baylor College of Medicine (SCP), Supported by NIH EY11915, NEI/NIH Core Grant EY-002520, Research to Prevent Blindness, The Oshman Foundation, William Stamps Farish Fund and the Hamill Foundation.

Purpose :
To evaluate the effects of cholinergic stimulation/blockade on distribution, dendritic cell (DC) uptake and immune response to antigen (Ag) applied to the mouse conjunctiva.

Methods :
Experiments were performed in 8-week old female C57/BL6 (B6) mice without and with pre-topical cholinergic stimulation with carbachol (CCh) or blockade with scopolamine (Scop). Distribution and DC uptake of topically applied FITC-labeled 2.3 kDA OVA323-339 peptide was evaluated by confocal microscopy in whole mount conjunctiva labeled with mucin or DC markers. OVA+ DCs were detected in the cervical lymph nodes (CLN) by flow cytometry 24 hr after topical instillation and compared to water controls. Proliferative response to topically applied OVA peptide (daily x 3 days) in the CLNs of OT-II (H-2b) transgenic mice was evaluated by WST assay.

Results :
CCh stimulation prior to topical OVA peptide application increased epithelial mucin binding. In contrast, cholinergic blockade with Scop caused diffuse paracellular antigen migration into the stroma. There were few OVA+CD11c+ or OVA+CD11b+ DCs in the epithelium or stroma under homeostatic conditions. After CCh stimulation, weakly OVA+ CD11c+ and OVA+CD11b+ cells were noted in the superficial stroma. Following Scop, strongly OVA+ CD11c+ and CD11b+ cells were present in the epithelium and stroma. In the cervical nodes, OVA+CD11c+ cells were non detectable in the water and OVA peptide groups and detectable in a low percentage of cells in the other groups (CCh+OVA = 0.04%, Scop+OVA = 0.1%). A greater percentage of OVA+CD11b+ cells were found in all groups, ranging from 0.11 (OVA) to 0.32 (Scop+OVA), with no statistical difference between groups. The percentage OVA+CD11b+CD11c+ cells was greater in the Scop+OVA group than the OVA and CCh+OVA groups (P ≤ 0.05). The number of proliferating cells in the CLNs following topical Ag challenge was lower in the OVA and CCh+OVA groups than the water control, while there was significantly higher proliferation (P ≤ 0.05) in the Scop+OVA than the other groups.